tbuckley/cabot-search huggingface dataset:
Abstract P3184: Catalase Knockdown Reverses The Activation Of Akt And The Increase In Mitochondrial Complex I Induced By MnSOD Overexpression In Mouse Heart Endothelial Cells
Authors: Rayane Brinck Teixeira, Cynthia M Xu, Christopher Chang, Frank W Sellke, Ruhul Abid
Journal: Circulation Research (2023)

Introduction: We have recently showed that endothelial cell (EC)-specific overexpression of mitochondrial antioxidant MnSOD (MnSOD-OE) induced Akt and ERK activation, mitochondrial complex I (C-I) biogenesis, supercomplex assembly, and a shift from glycolysis to mitochondrial respiration in mouse heart ECs (MHEC). In vivo, MnSOD-OE induced coronary angiogenesis and improved cardiac function in a transgenic animal model of myocardial infarction. Hypothesis: We hypothesize that antioxidant catalase mediated neutralization of MnSOD-induced conversion of superoxide to H 2 O 2 within mitochondria (mito) is crucial for C-I biogenesis, ATP synthesis, and Akt and ERK activation in MnSOD-OE MHEC. Methods: We used an EC-specific, tetracycline (Tet)-controlled, transgenic MnSOD-OE model. Isolated MHECs (Tet-ON=Control, Tet-OFF=MnSOD-OE) were reverse transfected with either negative control (NC)- or catalase-siRNA (Tet-ON NC or CAT siRNA, Tet-OFF NC or CAT siRNA) and evaluated by western blot and ATP luminescence. Data were analyzed by Two-Way ANOVA and Tukey’s post-hoc. A p -value ≤0.05 was considered statistically significant. Results: Catalase-siRNA resulted in 54% decrease in catalase expression (2.01±0.05 in Tet-OFF NC vs. 0.93±0.05 in Tet-OFF CAT siRNA) in MHEC, resulting in 69% increase in mito-ROS (17.33±1.71 in Tet-OFF NC vs. 29.33±2.42 in Tet-OFF CAT siRNA). Catalase knockdown and increased mito-ROS resulted in 30% decrease in p-44/42 ERK (1.92±0.08 in Tet-OFF NC vs 1.35±0.17 in Tet-OFF CAT siRNA) and 17% decrease in p-Akt ser473 /t-Akt ratio in MnSOD-OE MHECs (1.57±0.07 in Tet-OFF NC vs 1.30±0.08 in Tet-OFF CAT siRNA). MnSOD-OE MHECs also showed decreased expression of mitochondrial C-l (by 31%; 1.43±0.06 in Tet-OFF NC vs 0.99±0.03 in Tet-OFF CAT siRNA) and C-III (by 29%; 0.90±0.06 in Tet-OFF NC vs 0.64±0.03 in Tet-OFF CAT siRNA), and decreased ATP luminescence when compared with the control group (by 27%; 12836±568.6 in Tet-ON CAT siRNA vs 9319±650.8 in Tet-OFF CAT siRNA). Conclusions: Catalase appears to play crucial role in reducing mito-ROS and inducing C-I biogenesis, ATP synthesis, and activation of Akt and ERK in MnSOD-OE MHECs.
Abstract P2009: Mice Lacking S-nitrosoglutathione Reductase (gsnor <sup>-/-</sup> ), A Mouse Model Of Preeclampsia, Exhibit Abnormal Calcium Regulation And Cardiac Hypertrophy During Pregnancy.
Authors: Raul A Dulce, Wayne Balkan, Joshua M Hare, Shathiyah Kulandavelu
Journal: Circulation Research (2023)

Introduction: We recently showed that mice lacking S-nitrosoglutathione reductase (GSNOR -/- mice), a denitrosylase that regulates protein S-nitrosylation, exhibit the clinical features of preeclampsia (PE) a leading cause of maternal mortality. PE predisposes the mother to increased risk of cardiovascular (CV) injury later in life and is linked to cardiac hypertrophy and impaired relaxation. Cardiac hypertrophy and relaxation are regulated by calcium cycling. We hypothesized that GSNOR deficiency in pregnant mice alters calcium cycling, leading to hypertrophy and impaired cardiac relaxation. Methods: Pregnant control (WT [C57B6]) and GSNOR -/- mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) (N=3-4 mice/group; n=8-24 CMs/heart) were isolated and assessed for intracellular calcium and sarcomere shortening using IonOptix system. Dynamic parameters associated with calcium decay and sarcomere relaxation were recorded (time to peak (ttp), time to 50% of baseline (tt50%); d SL/ d t min , time to d [Ca]/ d t min , etc.). Experiments were conducted at 37°C. Results: At late pregnancy, systolic calcium levels were significantly elevated in CMs isolated from GSNOR -/- as compared to WT mothers. Despite the increased cytosolic calcium, CMs from pregnant GSNOR -/- mice exhibited no change in contractility. The improved calcium decay rate observed in CMs from pregnant versus NP WT mice, was absent in GSNOR -/- mothers. Similarly, sarcomere relaxation parameters and sarcomere resting length, which were also improved by pregnancy in WT CMs, were impaired in CMs from pregnant versus NP GSNOR -/- mice. In addition, hearts were significantly enlarged in GSNOR -/- mothers as compared to WT, even when normalized to tibia length. Conclusion: The elevated systolic calcium levels, impaired cardiac relaxation, and cardiac hypertrophy seen in pregnant GSNOR -/- mice may predisposes them to higher risk for CV injury during pregnancy and later in life.
Abstract P2187: SARS-CoV2 Nonstructural Protein, But Not The Omicron Ba.4/5 Mutant, Suppresses YAP Transcriptional Activity In The Heart
Authors: Fansen Meng, Chang-Ru Tsai, Jong H Kim, Xiao Li, Jeff Steimle, Shijie Liu, Bing Xie, Rich Gang Li, James F Martin
Journal: Circulation Research (2023)

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV2, is a worldwide epidemic resulting in global healthcare crises and strained health resources. Currently, the omicron subvariants of SARS-CoV2, BA.4 and BA.5, have become the predominant variant in circulation around the world. Compared to previous variants, omicron BA.4 and BA.5 escape antibodies created by past COVID-19 vaccines and prior infections due to spike protein mutations. However, why omicron subvariants induce milder symptoms after infection are poorly understood. Here we screened SARS-CoV2 proteins and found that one nonstructural protein suppresses the transcriptional activity of YAP, the Hippo pathway signaling effector, independent of negative regulation by upstream Lats kinases in the heart. Our findings correlate with RNA sequencing data from COVID-19 patient lung samples and iPS cardiomyocytes showing decreased expression levels of YAP target genes, which suggested that SARS-CoV2 infection may affect lung epithelial cell and neonatal cardiomyocyte proliferation. Additionally, we found that the Omicron BA.4/5 variant of this viral protein suppresses YAP activity significantly less compared to the wildtype protein whose mechanism are under exploring now. These findings reveal the previously unknown function and mechanism of SARS-CoV2 nonstructural proteins in host cells. Considering the critical role of Hippo signaling in cell proliferation, apoptosis, and immune response, our data on SARS-CoV2 protein function may partially explain the lesser toxicity of Omicron BA.4/5 to the host cells.
Abstract P3082: Loss Of Myeloid Cell-specific Beta2-adrenergic Receptor Improves Cardiac Function After Acute Ischemia
Authors: Tapas K Nayak, Anamika Bajpai, Rhonda Carter, Viren Patwa, Erhe Gao, Douglas G Tilley
Journal: Circulation Research (2023)

Heart failure (HF) is one of the leading causes of global mortality, which is often caused by acute myocardial infarction (MI). This type of acute cardiac injury promotes robust inflammatory cascades by regulating spatiotemporal myeloid cell (neutrophil, monocyte and macrophage) responses in the infarct area, which are essential in the orchestration of post-infarct tissue remodeling and repair. We previously reported that deletion of β2-adrenergic receptor (β2AR) in all cells of hematopoietic origin resulted in generalized disruption of immune cell responsiveness to injury, but with unknown impact of myeloid cells in particular. To investigate this, we crossed floxed β2AR (β2 f/f ) mice with myeloid cell-expressing Cre (LysM-Cre) mice to generate myeloid cell-specific β2AR knockout mice (LB2) and subjected them to MI. M-mode echocardiography indicated a preservation of ejection fraction (%EF) and fractional shortening (%FS) in the LB2 mice versus either β2 f/f or LysM-Cre mice at 3 days post-MI, which was maintained at 28 days post-MI, concurrent with reduced infarct size and interstitial fibrosis, suggesting a cardioprotective role of myeloid cell-specific β2-AR deletion. While immune cell profiling by FACS analysis showed similar CD45 + CD11b + Ly6C + monocyte, CD45 + CD11b + Ly6G + neutrophil and CD45 + CD11b + F4/80 + macrophage accumulation in LB2 and β2 f/f mouse hearts at 1 day post-MI, neutrophil frequency was significantly reduced in LB2 mouse hearts by 4 days post-MI, suggesting LB2 hearts have enhanced capacity for efferocytosis of dead cells. Notably, RT-qPCR analysis revealed a significant upregulation of pro-efferocytosis genes in LB2 bone marrow cells, including annexin A1 (ANXA1). Additionally, ANXA1 protein expression was shown to be upregulated in LB2 versus β2 f/f cardiac CD11b + myeloid cells, including macrophages, at 1-day post-MI. In vitro efferocytosis assay data suggested that there was significant increment in %-efferocytosis of LB2 neutrophils as compared to β2 f/f . Taken together, our data suggest that β2AR is a crucial regulator of myeloid cell-dependent efferocytosis in the heart following injury and suggests a crucial timepoint at which β2AR-selective therapeutics may promote the clearance of dead cells to promote repair.
Abstract P3167: Identification Of Macrophage-specific Markers On Extracellular Vesicles In A Transgenic Mouse Model And Humans
Authors: Natacha Carnel-Amar, Andras Saftics, Sarah Abu-Elreich, Michail Spanos, Emeli Chatterjee, Worawan Limpitikul, Ionita Ghiran, Stephen Gould, Robert Raffai, kendall Van Keuren-Jensen, Tijana Jovanovic-Talisman, Saumya Das
Journal: Circulation Research (2023)

Extracellular Vesicles (EVs) are key mediators of intercellular communication. Macrophages, a prominent type of immune cells, are known to release EVs with distinct contents and functions. The ExoMap mice model provides a platform to investigate tissue-specific EV subpopulations and the complexity of EV heterogeneity. CD169 and CD68 markers are specific to macrophages and have been associated with immune diseases, including Sarcoidosis. Double Transgenic vav-cre/ExoMap mice and appropriate controls were used to assay for immune cell derived EVs. Expression of cre in the vav-expressing cells, leads to expression of humanized CD81 (hsCD81) and mNeonGreen in the immune cells and in their EVs. Size Exclusion Column and immuno-pulldown (IP) with anti-hsCD81 or anti-CD169/CD68 antibodies were used to isolate macrophage-specific EVs. 3 miRNAs (mir223, mir374c, mir412) specific to macrophages were tested using Droplet Digital PCR (ddPCR). To design our human pilot study, we chose patients with active sarcoidosis (n=10), latent sarcoidosis (n=10), healthy controls (n=10) to compare the level of expression of specific immune markers in their blood. Bone marrow cells prepared from vav-cre/ExoMap mice were found to express mNeon Green and hsCD81. IP combined with vesicle flow cytometry defined the presence of hsCD81/mNeonGreen EVs in the plasma of double transgenic mice. Expression of CD68 and CD169 was robust on the EVs isolated using IP with hsCD81 antibodies from Double Transgenic mice. These results are consistent with the immune specificity of the vav-cre expression. Using human pooled plasma, we detected expression of CD68 on the EVs isolated using IP with CD169 antibody, suggesting the presence of both markers on macrophage specific EVs. Our findings support these markers as conserved between mouse and human samples, indicating their potential as universal markers of macrophage-derived EVs. Ongoing experiments aim to comprehensively characterize individual macrophage-derived EVs using the quantitative single molecule localization microscopy (qSMLM). Together, our findings provide a valuable resource for future studies of macrophage-EV communication and could serve in the development of EV-based diagnostic and therapeutic strategies.
Abstract P3049: Exploring The Therapeutic Potential Of Aav-mediated Restoration Of Col6a2 In A Mouse Model Of Ullrich Muscular Dystrophy
Authors: Julian R Havens, Vikram Prasad, Scott Blair, Jeff D Molkentin
Journal: Circulation Research (2023)

Ullrich muscular dystrophy is a genetic disease characterized by joint contractures and muscle weakness which leads to loss of ambulation and respiratory insufficiency early in life. This disease is caused by mutations in any of the three major genes of collagen VI - Col6a1, Col6a2, or Col6a3. Together, these three genes are required to generate a complex quaternary protein assembly that is secreted to help form the basement membrane. Currently there is no cure or greater treatment for Ullrich muscular dystrophy although 2 of the genes (Col6a1, Col6a2) are within the size limit of adeno-associated virus (AAV) and could be addressed by gene therapy. However, it is unclear what cell type in muscle generates the bulk of Collagen VI during development, or if it could be replaced at a later time by gene therapy from expression in myofibers or from fibroblasts. Given that mutations in the Col6a2 gene are the most common cause of Ullrich muscular dystrophy, here we generated Col6a2 gene deleted (Col6a2 -/- ) mice which showed the anticipated histopathologic evidence of dystrophic disease as young adults. Adult mice were then subject to retro-orbital injection of MyoAAV-Col6a2, which resulted in robust expression of Col6a2 within the myofibers of all skeletal muscles analyzed (and heart), although this was ineffective in restoring the collagen VI complex within the basement membrane. Further characterization into the effects of Col6a2 re-expression from the myofibers versus the fibroblasts in these gene targeted mice is underway with the goal of defining a combinatorial (with Col6a1 and Col6a3) or cell type restricted infection protocol to successfully achieve full restoration of Collagen VI in muscle.
Abstract P3191: Hur Mediates Calcium-driven Thermogenesis In Brown Adipose Tissue
Authors: Adrienne Guarnieri
Journal: Circulation Research (2023)

Since the discovery of brown adipose tissue (BAT) in adult humans, its high energy capacity has driven investigations targeting adipose tissue thermogenesis as a supplement to whole-body energy expenditure to mitigate the energy imbalances observed in obesity. Uncoupling protein 1 (UCP1) is the primary driver of thermogenesis, however several additional UCP1-independent pathways have been identified. We previously reported that adipocyte deletion of the RNA binding protein human antigen R (HuR) results in thermogenic dysfunction independent of UCP1. RNA sequencing revealed the downregulation of several genes involved in calcium ion transport upon HuR deletion. Although recently established as a thermogenic mechanism in beige adipocytes, the effects of calcium-mediated thermogenesis in brown fat and its contribution to whole-body metabolism remain unexplored. Therefore, we set out to investigate the impact and regulatory mechanism of HuR on SR calcium cycling and heat generation in BAT. We hypothesize that HuR regulates thermogenesis in brown adipose tissue through control of RyR-mediated SR calcium release and assessed this experimentally using murine models with a BAT-specific deletion of HuR. Brown adipocytes isolated from BAT-HuR -/- mice were imaged for cytosolic calcium and heat using Fluo-4 calcium indicator and ERthermAC dye respectively. Cohorts of mice were also subjected to acute cold challenge or placed on a 12-week high fat diet study to assess whole-body thermal and energy metabolism. We now show that loss of HuR decreases adrenergic-stimulated calcium release and heat generation in brown adipose tissue, and that this effect is rescued by pharmacological stabilization of RyR2. We also report that HuR directly binds RyR2 mRNA in vivo and loss of HuR reduces RyR2 mRNA transcript stability. In assessing whole-body metabolism, we observed impaired thermal tolerance of BAT-HuR -/- mice from controls, as well as sex-dependent effects on diet-induced weight gain between models. Our findings reveal, for the first time, that brown adipocytes participate in calcium-mediated thermogenesis and identify a regulatory role for HuR in coordinating heat production through maintenance of RyR2 calcium release activity.
Abstract P2024: Myofibroblast Ccn3 Contributes To Adverse Cardiac Outcomes Following Myocardial Infarction
Authors: Michael A Flinn, Santiago Alvarez, Makenna Knas, Caelan W Moreno, Sydney L Buday, Tyler Buddell, Reiauna Taylor, Jenny Drnevich, Michaela Patterson, Caitlin Omeara
Journal: Circulation Research (2023)

Myofibroblast function is critical for initial scar formation and prevention of catastrophic rupture following myocardial infarction, however, extended myofibroblast activity promotes pathological remodeling through exacerbated scar formation, myocyte uncoupling, and regulation of immune cells. Therefore, there exists a need to identify molecular mediators of myofibroblast activity to therapeutically target these pathways and regulate the fibrotic response following cardiac injury. It has been demonstrated the suppression of the transcription factors Yap and Wwtr1 dramatically reduces myofibroblast activity, but the mechanisms by which these Hippo pathway factors mediate pathological remodeling remain relatively unknown. Recently, we have characterized the secreted matrix protein Ccn3 as an element of pathological remodeling. Regulated in part by the Hippo pathway, fibroblast expression of Ccn3 increases shortly after infarction. Administration of recombinant human CCN3 dramatically promoted cardiopathology in adult mice following injury, resulting in exacerbated injuries, reduced cardiac function, and expression of the pro-fibrotic Tgfβ1 pathway. Treatment of cultured cardiac fibroblasts with CCN3 resulted in increased expression of the fibrotic factors such as Fn1, Serpine1, and Ccn2. Conversely, genetic knockout of Ccn3 specifically in myofibroblasts promoted improved cardiac function following infarction as compared to injured control mice. Thus, Ccn3 is an enticing target for therapeutic strategies to suppress pathological remodeling.
Abstract GS.101: Cardiomyocyte DCHS2 Inhibition Protects Against Heart Failure
Authors: Haobo Li, Jiayi Kang, CHUNYANG XIAO, JUSTIN GUERRA, Rebecca Freeman, James Rhee, Anthony Rosenzweig
Journal: Circulation Research (2023)

Loss of cardiomyocytes (CMs) and impaired mitochondrial function are major causes of heart failure (HF). We recently found that cardiac Dachsous Cadherin-Related 2 (DCHS2) is induced in pathological hypertrophy and HF but inhibited by exercise. CM DCHS2 expression in vivo causes pathological hypertrophy while its deletion induces physiological hypertrophy and increases markers of cardiomyogenesis. Whether and how DCHS2 inhibition protects against pathological hypertrophy or HF are unknown. CAS9 knock-in mice were injected with AAV9 carrying scrambled (control) or guide RNA1 (gRNA1) targeting DCHS2 exon 2 together with troponin T-driven Cre and subjected to transverse aortic construction (TAC). 10 weeks after TAC, DCHS2 knockdown (KD) in CMs reduced heart (HW) and lung weight / tibial length (TL) ratios (HW/TL: 11.14±0.76 vs 13.72±0.72 mg/mm, p=0.04, n=6/group), increased fractional shortening (40.67±3.65% vs 28.80±3.51%, p=0.04, n=6/group) and decreased CM size. DCHS2 KD in CMs increased markers of CM proliferation (Ki67 and pHH3, PCM1 double positive cells, all p&lt;0.05) and expression of mitochondrial respiratory chain complexes I-V protein and PGC1α mRNA (all p&lt;0.05) while attenuating TAC-induced disruption of mitochondrial structure and reactive oxygen species production measured by DHE staining. In primary CMs, DCHS2 gene KD by siRNA increased CM proliferation (13.93±1.09% vs 10.11±0.14%, p=0.03, n=6) which was prevented by Yap1 KD (13.93±1.09% in DCHS2 KD vs 7.45±0.89 in DCHS2/Yap1 double KD, p&lt;0.01, n=6). DCHS2 KD also increased mitochondrial oxygen consumption (354.8±25.86 vs 248.2±10.42 pmol/min, p&lt;0.01, n=6) and enhanced expression of mitochondrial biogenesis-related genes as well as mtDNA copy number (all p&lt;0.05). These DCHS2 KD-induced changes were not affected by Yap1 KD but were canceled by KD of either PGC1α or Acadvl, which is essential for very long chain fatty acids oxidation (all p&lt;0.05). DCHS2 pulldown demonstrated that Acadvl directly bound DHCS2 in CMs. In conclusion, DCHS2 inhibition protects against HF through Yap1-dependent cardiomyogenesis and Acadvl-dependent mitochondrial effects.
Abstract P1028: Elucidating Differential Mechanisms That Underlie Phenotypic Variability In Two Independent Dcm Models
Authors: Catherine Vasquez, Melissa L Lynn, Jil C Tardiff
Journal: Circulation Research (2023)

Mutations in components of the cardiac thin filament cause disease via complex, multi-faceted myocellular mechanisms, ultimately presenting as heterogeneous, symptomatic cardiomyopathies. The heterogeneity of both the myocellular mechanism(s) involved and the clinical outcome(s) represents a challenge in ongoing efforts to link genotype to phenotype. Two DCM-associated thin filament mutations (D230N-Tm and R173W-cTnT) with similar clinical phenotypes, were selected explore the molecular basis of disease trajectory. In patient populations, D230N-Tm exhibits an earlier time of onset compared to R173W-cTnT, with children (&lt;1yr) presenting with an often-fatal DCM. We hypothesize that the difference in time of onset can be explained by distinct myocellular mechanisms. We utilized transgenic mice expressing D230N-Tm and R173W-cTnT and defined the progression of pathogenic remodeling across 3 independent timepoints via 2D-echo. At 2 months the %FS of D230N-Tm was significantly reduced (21.79 ± 1.19) compared to NTg littermates (33.45±1.45). R173W-cTnT, however, exhibited no reduction in %FS until 4 months (22.53±0.78), thus phenocopying the patient population. Using these timepoints to guide our myocellular mechanics, contractility measurements were then utilized to assess the myocellular mechanics associated with the mutations. At 2 months D230N-Tm exhibited a decrease in the rate of contraction (4.53±0.24μm/sec) and rate of relaxation (2.88±0.23) compared to NTg. R173W-cTnT did not diverge from NTg until 4 months, exhibiting a later onset of impaired contractility. By 4 months, both mutations displayed the opposite, an increase in rate of contraction and relaxation (rate of contraction, D230N-Tm 4.49±0.18 and R173W-cTnT 5.83±0.27; and rate of relaxation, D230N-Tm 3.14±0.19 and R173W-cTnT 3.70±0.24). Our results suggest that the accelerated time course for the development of D230N-Tm – linked DCM is caused by differential myocellular mechanism(s) that induce the early reduction in contractility and relaxation observed. Ongoing studies aim to further identify possible myocellular mechanism(s) by which these mutations exhibit different myocellular mechanics.